Research

Dark Adaptation in Vertebrate Photoreceptors

The work in our laboratory is focused on understanding the response of the vertebrate eye to and recovery from the effects of bright light. Our principal approach is to make electrophysiological measurements of rod and cone photoreceptors of cold-blooded vertebrate animals, and to correlate these physiological responses to microspectrophotomeric measurements of the visual pigments as well as microfluorometric measurements of the concentration of vitamin A and Ca2+ contained within the cells. The reduction of all–trans retinal to Vitamin A is one of the principal initial steps that must occur following exposure to bright light to allow recovery of sensitivity (dark adaptation). Experiments have also shown that Ca2+ is a principal messenger substance during bright (bleaching) adaptation.

The Visual Cycle

Bleaching of rhodopsin (Rh) produces a photoactivated methrhodopsin II (R*), which decays through a series of intermediates to opsin and all-trans retinal. All-trans retinal is reduced within the photoreceptor to all-trans retinol, which is then transported via the interphotoreceptor retinol binding protein (IRBP) through the extracellular space to an adjacent layer of epithelial cells, called the retinal pigment epithelium (RPE). Within the RPE, the all-trans retinal is bound to cellular retinol binding protein (not shown) and then is esterified to fatty acits, mostly palmitic acid. The ester is converted by retinoid isomerase to 11-cis retinol, which is bound to yet another binding protein and oxidized by oxidoreductase to 11-cis retinal. Interphotoreceptor binding protein then returns the 11-cis retinal to the photreceptor, where it recombines with opsin to form dark adapted Rh.

Retinol Fluorescence

Our most recent work has focused on the time course of changes in retinol fluorescence intensity following a large bleach of the visual pigment. The bright field image at top left shows cellular fragments as well as one intact cone (lower left) and one intact red rod (upper right). The cells were suspended in a bath that had been mounted on the stage of the fluorescence microscope. The second field on the top row shows a fluorescence image obtained before visual pigment bleaching (T = 0.00 min). It is apparent that both intact cells exhibit a large amount of fluorescence in their ellipsoid regions. This fluorescence is consistent with the large concentration of mitochondria located there, and likely results from the high concentration of NADH in mitochondria. The cell was then exposed to sufficient bright green (500 nm) light to bleach in excess of 99% of the visual pigment contained in the outer segments of both cells. The top right panel (T = 0.52 min.) shows a large uniform increase in fluorescence in the region of the cone outer segment, and a small amount of fluorescence beginning to appear in the most proximal part of the outer segment of the rod. The bottom panel on the left (T = 13.09 min.) shows that by this time, much of the outer segment fluorescence in the cone had dissipated; however, the fluorescence in the rod outer segment continued to increase, and appeared to uniformly fill the outer segment of the rod. The image in the second panel from the left, bottom row (T = 37.80 min.) shows that the fluorescence in the cone outer segment by this time was very low, but that in the outer segment of the rod had achieved a maximum level. At this time 2 :m bovine IRBP was added to the bath. Thereafter, the fluorescence in the rod outer segment was observed to decline. The last measurement of fluorescence was made 87.82 min following the initial pigment bleach.